We investigated the effects of Flt3/Flk-2 ligand (FL) and interleukin-7 (IL-7) on DNA synthesis and proliferation of blast cells from patients with acute lymphoblastic leukemia (ALL). After 7 days of serum-free suspension culture of 19 samples, FL induced maximal DNA synthesis in two cases, whereas the combination of FL and IL-7 did so in another eight samples with a stimulation index (SI) >2. However, the number of viable cells after 7 days of liquid culture decreased in all but one sample. In this case of a pre-pre-B-ALL with a translocation t(4;11), FL induced dose-dependent proliferation (maximal 100 ng/mL) and cells stimulated with FL could be cultured for up to 4 weeks. A homogeneous population with 98% CD19-positive cells was detected before and after culture, and there was no evidence of nonleukemic cell proliferation as determined by immunophenotyping. The flt3 gene was transcribed in all seven cases studied by reverse-transcriptase polymerase chain reaction (RT-PCR). In the ALL cells responsive to FL, the expression of functional Flt3 receptors was confirmed by demonstrating FL-dependent tyrosine phosphorylation of Flt3. Furthermore, FL-dependent tyrosine phosphorylation of cellular proteins of estimated molecular weights of 70, 115, and 140 kD was detectable in these cells. These data demonstrate the functional heterogeneity of ALL samples and show that functional Flt3 receptors capable of mediating FL-dependent mitogenic signaling are expressed in a subset of ALL.